CRISPR/Cas9-mediated editing of Clpsk1 enhanced watermelon resistance to Fusarium oxysporum. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be an effective genome-editing tool for crop improvement. Previous studies described that Phytosulfokine (PSK) signalling attenuates plant immune response. In this work, we employed the CRISPR/Cas9 system to knockout Clpsk1 gene, encoding the PSK precursor, to confer enhanced watermelon resistance to Fusarium oxysporum f.sp. niveum (FON). Interactions between PSK and FON were analysed and it was found that transcript of Clpsk1 was significantly induced upon FON infection. Meanwhile, application of exogenous PSK increased the pathogen growth. Then, one sgRNA, which targeted the first exon of Clpsk1, was selected for construction of pRGEB32-CAS9-gRNA-Clpsk1 expression cassette. The construct was then transformed to watermelon through Agrobacterium tumefaciens-mediated transformation method. Six mutant plants were obtained and three types of mutations at the expected position were identified based on Sanger sequencing. Resistance evaluation indicated that Clpsk1 loss-of-function rendered watermelon seedlings more resistant to infection by FON. These results indicate that CRISPR/Cas9-mediated gene modification is an effective approach for watermelon improvement.

中文翻译：

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CRISPR/Cas9 介导的西瓜 Clpsk1 诱变赋予对尖孢镰刀菌 f.sp. 的抗性。妮维姆。

CRISPR/Cas9 介导的 Clpsk1 编辑增强了西瓜对尖孢镰刀菌的抗性。集群规则间隔短回文重复 (CRISPR)/Cas9 系统已被证明是一种有效的作物改良基因组编辑工具。先前的研究描述了植物硫素 (PSK) 信号传导减弱植物免疫反应。在这项工作中，我们使用 CRISPR/Cas9 系统敲除编码 PSK 前体的 Clpsk1 基因，以增强西瓜对尖孢镰刀菌的抗性。妮维姆 (FON)。分析了PSK和FON之间的相互作用，发现Clpsk1的转录物在FON感染后被显着诱导。同时，外源PSK的应用增加了病原体的生长。然后，一个 sgRNA，它针对 Clpsk1 的第一个外显子，选择用于构建 pRGEB32-CAS9-gRNA-Clpsk1 表达盒。然后通过根癌农杆菌介导的转化方法将该构建体转化为西瓜。获得了六株突变植物，基于Sanger测序鉴定了预期位置的三种突变类型。抗性评估表明，Clpsk1 功能丧失使西瓜幼苗对 FON 感染的抵抗力更强。这些结果表明，CRISPR/Cas9介导的基因修饰是西瓜改良的有效途径。抗性评估表明，Clpsk1 功能丧失使西瓜幼苗对 FON 感染的抵抗力更强。这些结果表明，CRISPR/Cas9介导的基因修饰是西瓜改良的有效途径。抗性评估表明，Clpsk1 功能丧失使西瓜幼苗对 FON 感染的抵抗力更强。这些结果表明，CRISPR/Cas9介导的基因修饰是西瓜改良的有效途径。